Newly synthesized eukaryotic mRNA molecules, also known as primary transcripts or pre-mRNA, made in the nucleus, are processed before or during transport to the cytoplasm for translation. Processing of the pre-mRNAs includes addition of a 5′ methylated cap and an approximately 200-250 base poly(A) tail to the 3′ end of the transcript.
Another step in mRNA processing is splicing of the pre-mRNA, which occurs in the maturation of 90-95% of mammalian mRNAs. Introns (or intervening sequences) are regions of a primary transcript (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions of a primary transcript that remain in the mature mRNA when it reaches the cytoplasm. The exons are spliced together to form the mature mRNA sequence. Splice junctions are also referred to as splice sites with the junction at the 5′ side of the intron often called the “5′ splice site,” or “splice donor site” and the junction at the 3′ side of the intron called the “3′ splice site” or “splice acceptor site.” In splicing, the 3′ end of an upstream exon is joined to the 5′ end of the downstream exon. Thus the unspliced RNA (or pre-mRNA) has an exon/intron junction at the 5′ end of an intron and an intron/exon junction at the 3′ end of an intron. After the intron is removed, the exons are contiguous at what is sometimes referred to as the exon/exon junction or boundary in the mature mRNA. Cryptic splice sites are those that are not used in wild-type pre-mRNA, but may be used when the natural splice site is inactivated or weakened by mutation, or in conjunction with a mutation that creates a new splice site elsewhere on the pre-mRNA. Alternative splicing, defined as the splicing together of different combinations of exons or exon segments, often results in multiple mature mRNA transcripts expressed from a single gene.
Up to 50% of human genetic diseases resulting from a point mutation are caused by aberrant splicing. Such point mutations can either disrupt a current splice site or create a new splice site, resulting in mRNA transcripts comprised of a different combination of exons or with deletions in exons. Point mutations also can result in activation of a cryptic splice site(s), disrupt a branch site (which functions during an intermediate step in splicing catalysis) or disrupt regulatory cis elements (i.e., splicing enhancers or silencers, which can be created, destroyed, strengthened or weakened by mutation) (Cartegni et al., Nat. Rev. Genet., 2002, 3, 285-298; Crawczak et al., Hum. Genet., 1992, 90, 41-54).
Antisense oligonucleotides have been used to target mutations that lead to aberrant splicing in several genetic diseases in order to redirect splicing to give a desired splice product (Kole, Acta Biochimica Polonica, 1997, 44, 231-238). Such diseases include β-thalassemia (Dominski and Kole, Proc. Natl. Acad. Sci. USA, 1993, 90, 8673-8677; Sierakowska et al., Nucleosides & Nucleotides, 1997, 16, 1173-1182; Sierakowska et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 12840-44; Lacerra et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 9591-9596); dystrophy Kobe (Takeshima et al., J. Clin. Invest., 1995, 95, 515-520); Duchenne muscular dystrophy (Dunckley et al. Nucleosides & Nucleotides, 1997, 16, 1665-1668; Dunckley et al. Human Mol. Genetics, 1998, 5, 1083-90); osteogenesis imperfecta (Wang and Marini, J. Clin Invest., 1996, 97, 448-454); and cystic fibrosis (Friedman et al., J. Biol. Chem., 1999, 274, 36193-36199).
Antisense compounds have also been used to alter the ratio of the long and short forms of Bcl-x pre-mRNA (U.S. Pat. Nos. 6,172,216; 6,214,986; Taylor et al., Nat. Biotechnol. 1999, 17, 1097-1100) or to force skipping of specific exons containing premature termination codons (Wilton et al., Neuromuscul. Disord., 1999, 9, 330-338). U.S. Pat. No. 5,627,274 and WO 94/26887 disclose compositions and methods for combating aberrant splicing in a pre-mRNA molecule containing a mutation using antisense oligonucleotides which do not activate RNAse H.
Antisense compounds targeting splicing-inhibitory elements in exons or their flanking introns have also been used to increase the use of such exons during splicing, e.g., in the context of spinal muscular atrophy (Cartegni Nat Struct Biol; Imaizumi; Hua PLoS Biol; Singh; other Hua et al papers, etc.).
Familial dysautonomia (FD), a rare genetic disorder found almost exclusively in the Ashkenazi Jewish population, is an autosomal recessive condition that is caused by a single intronic point mutation in intron 20 (IVS20+6T→C) of the IKBKAP gene (Maayan, C., Kaplan, E., Shachar, S., Peleg, O., and Godfrey, S. 1987, “Incidence of familial dysautonomia in Israel 1977-1981,” Clin Genet 32:106-108; Slaugenhaupt, S. A., and Gusella, J. F. 2002, “Familial dysautonomia,” Curr Opin Genet Dev 12:307-311; Anderson, S. L., Coli, R., Daly, I. W., Kichula, E. A., Rork, M. J., Volpi, S. A., Ekstein, J., and Rubin, B. Y. 2001, “Familial dysautonomia is caused by mutations of the IKAP gene,” Am J Hum Genet 68:753-758). FD, also known as Riley-Day syndrome and hereditary sensory autonomic neuropathy type-III (HSAN-III), is characterized by poor development and progressive degeneration of sensory and autonomic neurons. Notable symptoms include anhidrosis, decreased taste, depressed deep tendon reflexes, postural hypotension, loss of pain and temperature perception, alacrima, gastroesophageal reflux, and scoliosis (Axelrod, F. B., and Simson, G. G. V. 2007 “Hereditary sensory and autonomic neuropathies: types II, III, and IV,” Orphanet Journal of Rare Diseases 2:). The extent and severity of the symptoms vary among patients, but even with advanced management, the disease leads to premature death, with only half of the patients surviving to 40 years of age.
Antisense technology is an effective means for modulating the expression of one or more specific gene products, including alternative splice products, and is uniquely useful in a number of therapeutic, diagnostic, and research applications. The principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities, such as transcription, splicing or translation, through one of a number of antisense mechanisms. The sequence specificity of antisense compounds makes them extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in disease.